Discovery of Molecular Switches That Modulate Modes of

Jun 19, 2009 - Discovery of Molecular Switches That Modulate Modes of Metabotropic Glutamate Receptor Subtype 5 (mGlu5) Pharmacology in Vitro and in ...
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J. Med. Chem. 2009, 52, 4103–4106 4103 DOI: 10.1021/jm900654c

Discovery of Molecular Switches That Modulate Modes of Metabotropic Glutamate Receptor Subtype 5 (mGlu5) Pharmacology in Vitro and in Vivo within a Series of Functionalized, Regioisomeric 2- and 5-(Phenylethynyl) pyrimidines Sameer Sharma,† Jeffrey Kedrowski,‡ Jerri M. Rook,‡ Randy L. Smith,‡ Carrie K. Jones,‡,§ Alice L. Rodriguez,‡,§ P. Jeffrey Conn,‡,§ and Craig W. Lindsley*,†,‡,§ †

Department of Chemistry, ‡Department of Pharmacology, and Vanderbilt Program in Drug Discovery, Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee 37232 §

Received May 18, 2009 Abstract: We describe the synthesis and SAR of a series of analogues of the mGlu5 partial antagonist 5-(phenylethynyl)pyrimidine. New molecular switches are identified that modulate the pharmacological activity of the lead compound. Slight structural modifications around the proximal pyrimidine ring change activity of the partial antagonist lead to that of potent and selective full negative allosteric modulators and positive allosteric modulators, which demonstrate in vivo efficacy in rodent models for anxiolytic and antipsychotic activity, respectively.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and exerts its effects through both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRsa) are members of the G-protein-coupled recpetor (GPCR) family C, which are characterized by a large extracellular aminoterminal agonist-binding domain. To date, eight mGluRs have been cloned, sequenced, and assigned to three groups (group I, mGlu1 and mGlu5; group II, mGlu2 and mGlu3; group III, mGlu4,6,7,8) based on their sequence homology, pharmacology, and coupling to effector mechanisms.1,2 In preclinical models, studies with the negative allosteric modulators (NAMs) 1 (MPEP) and 2 (MTEP) (Chart 1) have demonstrated that selective antagonism of mGlu5 has therapeutic potential for chronic disorders such as pain, anxiety, depression, addiction, and fragile X syndrome.3-7 Furthermore, there is direct clinical validation of anxiolytic activity by allosteric antagonism of mGlu5 in patients with fenobam 3.8 Alternatively receptor activity can be enhanced through positive allosteric modulators (PAMs) such as 4 (DFB), 5 (CPPHA), 6 (CDPPB), and 7 (ADX-47273), which with the exception of 5 share the same allosteric binding site as 1.9-13 PAMs 6 and 7, both ago-potentiators, have demonstrated in vivo proof of concept in preclinical schizophrenia models in which other known antipsychotics show similar positive effects.10-13 Recently, pure mGlu5 PAMs have been devel-

oped based on 7, by the incorporation of a basic heterocycle in the 3-position of the oxadiazole.14 On the basis of our experience in the development of allosteric modulators of mGluRs with a broad range of activities including negative allosteric modulators, positive allosteric modulators and neutral allosteric site ligands at the allosteric binding site occupied by 1, together with theoretical models of allosteric function, we postulated that it might be possible to develop “partial antagonists”. As envisioned, a “partial antagonist” would fully occupy the binding site of 1 on the mGlu5 receptor but only partially block agonist response, resulting in partial mGlu5 inhibition; moreover, Rodriguez et al. identified several mGlu5 partial antagonists.15 In 2008, Sharma et al. conducted a limited optimization effort focused on the mGlu5 partial antagonist lead 8. Within two 24-member libraries, SAR elucidated a “molecular switch” to modulate pharmacological activity (Figure 1).16 Lead 8, with an unsubstituted distal phenyl ring, fully occupied the allosteric binding site of 1, possessed an IC50 of 486 nM, but only afforded partial response (29% response, 71% partial antagonism), that is, allosteric partial antagonism. Incorporation of small chemical moieties in the 3-position of the distal phenyl ring, such as a 3-methyl group, delivered 9, a full noncompetitive mGlu5 antagonist (IC50 = 7.5 nM). When the methyl group was moved from the 3-position to the 4-position as in 10, an efficacious (99% of glutamate max) mGlu5 PAM resulted (EC50=3.3 μM, 4.2-fold shift), which also represented a new mGlu5 PAM chemotype.16 The observation of a conserved molecular switch, accessed by toggling between 3- and 4-substitution on the distal phenyl ring, within this chemical series was unprecedented. These preliminary data encouraged us to further optimize 8 and survey the impact of incorporating incorporating substituents on the pyrimidine ring, as well as examining regioisomeric pyrimidines to develop potent and Chart 1. mGlu5 Allosteric Ligands

*To whom correspondence should be addressed. Phone: 615-3228700. Fax: 615-322-8577. E-mail: [email protected]. a Abbreviations: mGluR, metabotropic glutamate receptor; NAM, negative allosteric modulator; PAM, positive allosteric modulator; GPCR, G-protein-coupled receptor. r 2009 American Chemical Society

Published on Web 06/19/2009

pubs.acs.org/jmc

4104 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

selective mGlu5 NAMs and PAMs suitable for in vivo studies to confirm the observed in vitro pharmacology. For the next round of chemical lead optimization, we relied on an iterative analogue library synthesis approach17 to rapidly prepare a 24-member library18 in which 2-substituted-5-bromopyrimidines 11 were treated with phenylacetylene 13, 3-methylphenyl acetylene (the NAM “switch”) 14, or 4-methylphenyl acetylene (the PAM “switch”) 15 under

Figure 1. Identification of “molecular switches” that convert an mGlu5 partial antagonist 8 to a full noncompetitive antagonist (NAM) 9 or a weak but fully efficacious mGlu5 positive allosteric modulator (PAM) 10.

Scheme 1. Synthesis of Analogues of 16 and 17a

a Reagents and conditions: (a) 10 mol % Pd(PPh3)4, 20 mol % CuI, 20.0 equiv of diethylamine, DMF, microwave, 70 °C, 10 min, 16-95%; all compounds purified by mass-directed HPLC to >98% purity.19

Sharma et al.

microwave-assisted Sonogashira conditions (Scheme 1) to provide analogues 16. In parallel, we prepared a small threemember library employing the regiosiomeric 2-bromopyrimidine 12 and 13-15 to deliver analogues 17. SAR from this library was “flat”, with few actives (Table 1); however, unexpected modulation of the mode of mGlu5 pharmacology was observed. All new analogues 16 containing the 4-methylphenyl moiety were uniformly inactive, save for 16f, a weak mGlu5 PAM. When R1 was an ethoxy group in combination with the NAM “switch”, 3-methylphenyl, 16a resulted, a potent mGlu5 NAM (IC50 = 21 nM). The remaining analogues 16 were inactive or, more surprisingly, potent mGlu5 PAMs. When an aminomethyl group was incorporated at the 2-position of the pyrimidine, in conjunction with an unsubstituted phenyl ring, 16b resulted, which represents the most potent (EC50 = 14.3 nM, 15-fold shift) rat mGlu5 PAM reported to date (10- to 15-fold more potent than 6 and 7). Addition of the NAM “switch” 3-methylphenyl moiety with the 2-aminomethyl group 16c unexpectedly afforded a similarly potent mGlu5 PAM (EC50 = 21.1 nM, 5.9-fold shift), suggesting the 3-methylphenyl moiety is not a conserved molecular switch for engendering NAM activity. Interestingly, the NAM 16a differs from the PAM 16c by substitution at the 2-position of the pyrimidine, OEt versus NHMe, respectively, with equal potency (IC50 =21 nM and EC50=21 nM, respectively) but opposite mode of pharmacology. Other groups were tolerated in the 2-position of the pyrimidine such as SMe 16d and t-Bu 16e and found to engender mGlu5 PAM activity (EC50 = 120 nM, 11-fold shift and EC50 = 247 nM, 6-fold shift, respectively) but were inactive in the presence of the 3- or 4-Me-phenyl moieties. Overall, 16b represents a 235-fold improvement in potency over mGlu5 PAM 10, was selective for mGlu5 (>10 μM vs mGlu1-4,7,8), and warranted further evaluation. The PAMs reported here demonstrated no activity in the absence of glutamate, but in the presence of a subthreshold concentration of glutamate (EC20), a concentration dependent potentiation of mGlu5 response was observed (Figure 2). Importantly, 16b is a pure mGlu5 PAM, not an ago-potentiator like 6 and 7. In addition, 16b demonstrated a robust 15-fold leftward shift of the glutamate concentration response

Table 1. Structures, Activity, and Mode of Pharmacology of Analogues 16 and 17

8 9 10 16a 16b 16c 16d 16e 16f 17a 17b 17c

R1

R2

allosteric activitya

IC50, EC50 (nM)a

antagonism (%)a

fold shifta

H H H OEt NHMe NHMe SMe t-Bu NHMe N/A N/A N/A

H 3-Me 4-Me 3-Me H 3-Me H H 4-Me H 3-Me 4-Me

PA NAM PAM NAM PAM PAM PAM PAM PAM NAM NAM N/A

486 ( 28 7.5 ( 1.2 3,300 ( 290 21.1 ( 2.8 14.3 ( 2.3 21.1 ( 1.8 120 ( 25 247 ( 24 704 ( 86 195 ( 65 10.8 ( 2.1 >10000

71 100 N/A 100 N/A N/A N/A N/A N/A 100 100 N/A

N/A N/A 3.3 N/A 15 5.9 11 6.0 5.7 N/A N/A N/A

a IC50, EC50, antagonism, and fold shift are the average of at least three independent determinations. N/A = not applicable. PA = partial antagonist. NAM = negative allosteric modualtor. PAM = positive allosteric modulator. Fold shift at 10 μM fixed concentration of compound.

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Figure 3. Reversal of amphetamine-induced hyperlocomotion with mGlu5 PAM 16b in dose-dependent manner with the nontoxic vehicle, 10% Tween-80.

Figure 2. Compound 16b potentiates mGlu5 activation by glutamate. In the absence of glutamate, 16b does not activate mGlu5. In the presence of a subthreshold quantity of glutamate, 16b potentiates mGlu5 in a concentration-dependent manner. Compound 16b’s potentiation of response to glutamate is manifested as increased mGlu5 agonist sensitivity. The glutamate EC50 is shifted from 493 to 32 nM, or a 15-fold shift with 10 μM 16b.

curve (EC50 shifts from 493 to 32 nM) with an increase in glutamate max (Figure 2). In the regiosiomeric pyrimidine series 17, the 4-Me congener 17c was inactive. The unsubstitiuted phenyl analogue 17a was a moderately potent mGlu5 NAM (IC50 =195 ( 65 nM). Unlike series 16, the 3-Me NAM “switch” performed as expected in series 17, significantly increasing mGlu5 NAM activity (IC50 =10.8 ( 2.7 nM) for 17b. Moreover, 17b was selective for mGlu5 (>10 μM vs mGlu1-4,7,8). With a potent mGlu5 PAM 16b and a potent mGlu5 NAM 17b, we were poised to determine if the modes of mGlu5 modulation observed in our in vitro cellular assays would be mirrored in standard in vivo behavioral paradigms. To evaluate the PAM 16b, we chose to study the ability of 16b to reverse amphetamine-induced hyperlocomotion in rats, as 6 and 7 displayed robust efficacy in this preclinical model where other known antipsychotic agents show similar positive results.10-13 In the event, 16b was dosed ip at 3, 10, or 30 mg/kg 30 min prior to sc administration of 1 mg/kg amphetamine. As shown in Figure 3, a modest dose response is observed with 16b, with significant reversal noted at the 30 mg/kg dose, and no effect (i.e, sedation) of 16b/vehicle alone. Thus, the mGlu5 PAM activity observed in cell-based in vitro assays is mirrored in vivo with 16b and comparable to the effects seen with 6 and 7.10-13 Moreover, the reversal of amphetamine-induced hyperlocomotion with 16b is important, as 16b lacks the intrinsic agonism of the ago-potentiators 6 and 7, suggesting for the first time that positive allosteric modulation alone is sufficient for an antipsychotic profile in this preclinical model. Previously, mGlu5 NAMs such as 1 and 2 have demonstrated anxiolytic activity in numerous preclinical models.

Figure 4. Dose-response curves for the effects of 17b on punished (upper panel) and unpunished (lower panel) responding. The data are the mean number of punished and unpunished responses that animals made when tested on 1, 3, 10, 30 mg/kg 17b and vehicle. Each value represents the mean ( SEM for 18 animals. For punished responding animals tested on 10 and 30 mg/kg 17b made significantly greater number of responses than animals tested on vehicle (p < 0.05). Unpunished responding did not change significantly at any of the doses tested.

Therefore, 17b was tested in a modified Geller-Seifter conflict model wherein an increase in punished responding is consistent with an anxiolytic-like profile.20 As seen in Figure 4, 17b produced a significant dose-dependent increase in punished responding with the 30 mg/kg dose approaching a 300% increase in response rate [F(4,17) = 22.69, p < 0.0001] (upper panel) with no significant effect on unpunished responding (lower panel). Post hoc analysis indicated that the 10 and 30 mg/kg doses in the punished component of the schedule differed significantly from vehicle ( p